Growth and Welfare Status of Giant Freshwater Prawn (Macrobrachium rosenbergii) Post-Larvae Reared in Aquaponic Systems and Fed Diets including Enriched Black Soldier Fly (Hermetia illucens) Prepupae Meal

Simple Summary The giant freshwater prawn culture is still challenged by dependence on diets containing high percentages of unsustainable and expensive marine-derived ingredients that are necessary to ensure the proper growth and welfare of this species. The present study proposed the replacement of fish meal and fish oil with black soldier fly full-fat prepupae meal enriched with spirulina in order to formulate two experimental diets intended for giant freshwater prawn, which were tested during a 60-day feeding trial in aquaponic systems. The present research represents an example of implementing more sustainable aquafeeds and farming techniques that promote both the valorization of circular economies and zero-waste concepts. Abstract Due to the limited application of insect meal in giant freshwater prawn (Macrobrachium rosenbergii) culture, the present study aimed to (i) produce spirulina-enriched full-fat black soldier fly (Hermetia illucens) prepupae meal (HM) and (ii) test, for the first time, two experimental diets characterized by 3% or 20% of fish meal and fish oil replacement with full-fat HM (HM3 and HM20, respectively) on M. rosenbergii post-larvae during a 60-day feeding trial conducted in aquaponic systems. The experimental diets did not negatively affect survival rates or growth. The use of spirulina-enriched HM resulted in a progressive increase in α-tocopherol and carotenoids in HM3 and HM20 diets that possibly played a crucial role in preserving prawn muscle-quality traits. The massive presence of lipid droplets in R cells in all the experimental groups reflected a proper nutrient provision and evidenced the necessity to store energy for molting. The increased number of B cells in the HM3 and HM20 groups could be related to the different compositions of the lipid fraction among the experimental diets instead of a nutrient absorption impairment caused by chitin. Finally, the expression of the immune response and stress markers confirmed that the experimental diets did not affect the welfare status of M. rosenbergii post-larvae.


Introduction
The giant freshwater prawn Macrobrachium rosenbergii is one of the most cultured crustacean species worldwide due to its nutritional value, good meat quality, and high because they represent a feed supplement inclusion or an ecological inclusion level (a global 20% FM substitution in aquafeeds has a proper ecological impact), respectively. Particular emphasis was given to (i) growth performances and morphological indices, (ii) physical and chemical features of both muscle and exoskeleton, and (iii) the overall health status determined by the histological and molecular analyses on the hepatopancreas, which is the largest organ in the digestive tract of Decapoda and which is involved in absorption and metabolization of nutrients, as well as in the immune response [41]. To further improve prawn culture sustainability, a 60-day feeding trial was conducted in an aquaponic system, as different studies evidenced the suitability of this rearing method for M. rosenbergii culture [42,43].

Feeding Substrate Preparation and Insect Rearing
The feeding substrate of H. illucens larvae was mainly composed of coffee silverskin (a coffee industry by-product) prepared according to the method of Ratti et al. [33]. The coffee by-product was blended and reached a particle size of 0.4 ± 0.2 mm by using an Ariete 1769 food processor (De' Longhi Appliances Srl, Treviso, Italy). The feeding substrate (coffee by-product) for H. illucens larvae was prepared by including 15% (w/w) of dried Arthrospira platensis biomass and by adding distilled water to reach final moisture of~70% [44].
Six-day old H. illucens larvae (provided by Smart Bugs s.s., Ponzano Veneto, Treviso, Italy) were reared on the feeding substrate for 21 days in a climatic chamber in continuous darkness at 27 ± 1 • C, relative humidity of 65 ± 5%, and 0.3/cm 2 density [45,46]. The feeding rate was 100 mg/day for each larva [47], maintained by adding, weekly, a new feeding substrate. The transition to the prepupal stage was identified by the change in tegument color from white to black [48]. Prepupae were then collected, washed, freezedried, and stored at −80 • C.

Prawns' Diets Production
The freeze-dried full-fat H. illucens prepupae were ground to obtain fine powder for the prawns' diet preparation (Retsch Centrifugal Grinding Mill ZM 1000, Retsch GmbH, Haan, Germany). Three test diets were formulated, according to the report of Goda [49], to be grossly iso-proteic (36% dry matter, DM), iso-lipidic (9% DM), and isoenergetic (18 MJ/kg DM), as far as possible. Ingredients and proximate composition of the experimental diets used in the present study are reported in Table 1. A control diet (named HM0) containing fish meal (FM), vegetable-derived ingredients, and fish oil (FO) was prepared and used as the basal formulation. The test diets that included black soldier fly prepupae meal (HM) were prepared by replacing, from the HM0 formulation, 3% (HM3) and 20% (HM20) of marine-derived ingredients (both FM and FO) with full-fat HM, previously obtained.
The test diets were manufactured in the pilot feed mill plant at the Department of Agrifood, Environment and Animal Science of the University of Udine (Udine, Italy). All the ingredients were weighed and placed in a mixer; during mixing, water was gradually added (approximately 400 g/kg) to obtain a wet consistency forming dough. Then, the dough was cold extruded with a meat mincer (provided with a knife at the die) to obtain 2 mm pellets that were subsequently dried at 40 • C for 48 h in a ventilated oven. The diets obtained were stored in sealed bags under vacuum and kept at −20 • C until use.
The test diets were analyzed for proximate composition following AOAC [50] procedures. The measure of moisture content was carried out by weight loss after drying samples in an oven at 105 • C until a constant weight was reached. The incineration to a constant weight, conducted in a muffle oven at 450 • C (HD 230, AMSE s.r.l.; Torino, Italy), was used to evaluate the ash content. Crude protein was determined as total nitrogen (N) through the Kjeldahl method and multiplying N by 6.25, while crude lipids were determined according to the Soxhlet ether method. Gross energy was measured by a calorimetric bomb (Adiabatic Calorimetric Bomb Parr 1261; PARR Instrument, IL, USA). For the HM proximate composition (% DM), please refer to Ratti et al. [33]. The ingredients, the proximate composition, the fatty acids profile, and the α-tocopherol and carotenoids content (determined as previously described in Ratti et al. [33]) of the test diets used in the present study are reported in Table 1. Table 1. Ingredients (g/kg as a fed-basis), proximate composition (% on dry matter, DM), fatty acids profile, and α-tocopherol and carotenoid content of the test diets.

Aquaponic Systems and Experimental Design
One thousand nine hundred seventy-one giant freshwater prawn post-larvae (initial weight: 0.10 ± 0.01 g) were provided by Miami Aqua-culture Inc, (Miami, FL, USA) and acclimated for two weeks in a single 500 L tank (mechanical, biological, and UV filtration provided by Panaque, Viterbo, Italy) at the aquaponics facility (MJ Energy srl, Treia, Macerata, Italy). Tank temperature was 28.0 ± 0.5 • C; ammonia and nitrite were <0.05 mg/L and nitrate <10 mg/L. At the end of the acclimation period, the prawns were randomly divided into nine Media Based Aquaponic Systems (720 L each of total volume) according to the 3 dietary treatments (three systems per experimental group, 219 specimens per tank). Each system consisted of a 1.56 m 2 hydroponic unit for plant cultivation and a 600 L prawns' tank.
Hydroponic unit. In each hydroponic unit, before the introduction of prawns in the aquaponic system, 15 lettuce (Lactuca sativa) seedlings, with an initial weight of 5.14 ± 0.47 g, were planted (density = 10 plants/m 2 ). Expanded clay was added to each hydroponic unit to ensure both mechanical and biological filtration [51], and to guarantee physical support for plant growth. A 1900 L/h pump (Eheim GmbH & Co, Deizisau, Germany) was used to guarantee 3 water renewals per hour. From the prawns' unit, water was pumped to the hydroponic one, then returned through a siphon equipped with synthetic foam to ensure an additional mechanical filtration.
The feeding trial duration was 60 days, during which the prawns almost tripled their weight. The prawns were hand-fed the experimental diets, provided at a feeding rate of 10% body weight, according to Posadas [52] (divided into two equal amounts, one in the morning and one in the afternoon), as follows: (i) prawns fed the control diet (the HM0 group), and (ii) prawns fed diets characterized by 3% or 20% of FM and FO replacement with HM (the HM3 and HM20 groups, respectively). The daily feeding ration was computed every 2 weeks by weighing a representative number of specimens per tank The feed particle size was 0.3 mm (obtained by mincing and sieving the pellets extruded during the preparation of the test diets). At the end of the trial, the prawns were sacrificed by thermal shock for individual measurements and for the excision of the cephalo-thorax for collection of hepatopancreas samples (which were properly stored for further analyses).

Survival Rate, Growth Performances, and Morphological Features
At the end of the trial, all the prawns were counted and weighted to calculate survival rates (SRs) and growth performances (weight gain, WG; relative growth rate, RGR; specific growth rate, SGR), using the following formulae: For the morphological characterization, a different set of 100 prawns from each tank was randomly collected to (i) measure FBW, total length (TL), and muscle lengths (ML), and (ii) calculate the incidence of both muscle weight (MW) and the weight of head and exoskeleton (HEW) on the final body weight (FBW), as well as the condition factor (K), as follows: Incidence of MW = MW/FBW × 100

Histological Analysis
At the end of the trial, hepatopancreas samples were obtained from five prawns per tank (15 prawns per dietary group) and immediately fixed in Bouin's solution for 24 h at 4 • C, then washed with ethanol (70%) [53]. The samples were dehydrated in 80%, 95%, and 100% ethanol solutions, washed with xylene, and embedded in paraffin (Bio-Optica, Milano, Italy). The solidified paraffin blocks were cut with a Leica RM2125 RTS microtome (Leica, Nussloch, Germany) to obtain 5 µm sections that were subsequently stained with Mayer hematoxylin and eosin Y (Merck KGaA, Darmstadt, Germany). Stained sections were examined (×20 and ×40 magnification) using a Zeiss Axio Imager.A2 (Zeiss, Oberkochen, Germany) microscope equipped with a combined color digital camera (Axiocam 105, Zeiss, Oberkochen, Germany), and image analysis was performed using the ZEN 2.3 software (Zeiss, Oberkochen, Germany). Histological analyses were focused on the middle portion of each hepatopancreas tubule (called B cell zone), as this part includes the mature stage of B and R cells [41]. The relative abundance of B and R cells, as well as the abundance of lipid droplets in R cells (an indication of the degree of lipid accumulation), the tubule diameter, and the height of the epithelium, were measured on 20 randomly selected tubules per section (3 sections per prawn, 15 prawns per dietary treatment). The score-assignment criteria for B and R cells' relative abundance and R cells' vacuolization are reported in Table 2.

RNA Extraction and cDNA Synthesis
Total RNA was extracted from hepatopancreas samples of 5 prawns from each tank (15 prawns per dietary group) using RNAzol RT reagent (Merck KGaA, Darmstadt, Germany) and finally eluted in RNase-free water (Qiagen, Hilden, Germany; 20 µL) and stored at −80 • C [54]. The NanoPhotometer P-Class (Implen, München, Germany) was used to determine RNA concentration, while RNA integrity was checked by GelRed TM staining of 28S and 18S ribosomal RNA bands on 1% agarose gel. The iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) was used to synthesize the cDNA from 1 µg of RNA.

Real-Time PCR
PCRs were performed in an iQ5 iCycler thermal cycler (Bio-Rad, Milano, Italy) setting a 96-well plate. For each sample, reactions were set mixing 1 µL of 1:10 diluted-cDNA, 5 µL of fluorescent intercalating agent (2× concentrated iQ ™ Sybr Green, Bio-Rad, Milano, Italy) and 0.3 µM of both forward and reverse primer [55]. For all reactions, the thermal profile was 3 min at 95 • C, then 45 cycles of 20 s at 95 • C, 20 s at the annealing temperature, specific for each primer and reported in Table 3, and 20 s at 72 • C. In all cases, one single peak was detected in the melting curve analyses, no amplification products were observed in negative controls, and no primer-dimer formations were observed in control templates. The relative quantification of the expression of genes involved in molting regulation (juvenile hormone epoxide hydrolase, jheh), enzymatic hydrolysis of chitin (chitinase 3, chit3), protein digestion (cathepsin L, catL), stress response (heat shock protein 90, hsp90), and immune response (α2-macroglobulin, α2m) was performed. Beta-actin (β-actin) and 18S ribosomal protein (18s) were used as housekeeping genes. Amplification products were sequenced, and homology was verified. The iQ5 optical system software version 2.0 (Bio-Rad), including GeneEx Macro iQ5 Conversion and GeneEx Macro iQ5 files, was used to analyze PCR data. Table 3. Sequences, identification numbers, and annealing temperature (AT) of primers used in the present study.

Physical and Chemical Analyses
For each dietary group, 4 pools consisting of the homogenized muscle of 12 prawns were prepared. pH was measured with a pH-meter METTER TOLEDO (Metter-Toledo, Schwerzenbach, Switzerland). Moisture was calculated gravimetrically by freeze-drying each pool. Starting from the freeze-dried material, total lipids and fatty acid (FA) profiles were assessed, following Folch et al. [58]. The primary oxidative products (conjugated dienes, CD), as well as the secondary lipid oxidation products (thiobarbituric acid reactive substances, TBARS), were analyzed according to Srinivasan et al. [59] and Vyncke [60], respectively. Results were expressed as mmol hydroperoxides (mmol Hp) and malondialdehyde equivalents (MDA-eq.) on 100 g freeze-dried prawn muscle for CD and TBARS, respectively.
For each dietary group, 4 pools consisting of the homogenized exoskeleton of 12 prawns were prepared to analyze the chitin content, following Hahn et al. [61].

Statistical Analysis
The aquaponic systems were used as the experimental unit for data related to survival rates and growth performance, while individual specimens were the experimental units for all the remaining analyses. All data were checked for normality (Shapiro-Wilk test) and homoscedasticity (Levene's test). Data for survival rates, growth parameters, histological analysis, and relative quantification of gene expression were analyzed through one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison post hoc test, performed using the software package Prism 8 (GraphPad software version 8.0.2, San Diego, CA, USA). Significance was set at p < 0.05.
Data on morphological traits and on physical and chemical characteristics were assessed with a one-way ANOVA followed by a Tukey's test using the free software environment R [62], with significance set at p < 0.05. All data were expressed as mean ± standard deviation (SD).

Survival Rate, Growth Performance and Morphological Features
All the daily-provided feed was consumed by the prawns of each tank within 15 min. Survival rates, growth performance, and the morphological features of M. rosenbergii are reported in Table 4. No significant differences were evident among the experimental groups considering the indices calculated, except for HEW, for which prawns from the HM20 group exhibited a significantly higher value than those from both the HM0 and HM3 groups (p < 0.001). Values are shown as mean ± SD (n = 3 for survival rate and growth performance; and n = 100 for morphological features). a,b Different letters indicate significant differences among experimental groups (p < 0.05). Abbreviations: SR, survival rate; IBW, initial weight; FBW, final weight; WG, weight gain; RGR, relative growth rate; SGR, specific growth rate; MW, incidence of muscle weight on FBW; HEW, incidence of the weight of head and exoskeleton on FBW.

Histological Analysis
No histopathological alterations were evident in all the tubules analyzed. Hepatopancreas samples from all the experimental groups showed a well-organized tubular structure (Figures 1 and 2) with diffused R cells characterized by highly abundant lipid droplets (Table 5). However, higher number of B cells was detected in hepatopancreas samples from prawns fed the HM3 and HM20 diets, compared with those fed on the HM0 diet. No significant differences were evident among the experimental groups in terms of tubule diameter or epithelium height (Table 5).

Histological Analysis
No histopathological alterations were evident in all the tubules analyzed. Hepatopancreas samples from all the experimental groups showed a well-organized tubular structure (Figures 1 and 2) with diffused R cells characterized by highly abundant lipid droplets (Table 5). However, higher number of B cells was detected in hepatopancreas samples from prawns fed the HM3 and HM20 diets, compared with those fed on the HM0 diet. No significant differences were evident among the experimental groups in terms of tubule diameter or epithelium height (Table 5).       Values of tubule diameter and epithelium height are shown as mean ± SD (n = 15).

Gene Expression
As evidenced in Figure 3, no significant differences were evident among the experimental groups in terms of jheh, chit3, hsp90, or α2m gene expression. However, catL was significantly upregulated in the HM3 and HM20 groups, compared with the HM0 group (Figure 3c).

Physical and Chemical Analyses
No significant differences were detected among the experimental groups considering pH, moisture muscle samples, and chitin content of exoskeleton samples from prawns fed the different experimental diets (Table 6). Table 6. pH and moisture (g/100g of muscle) measured in the muscle and chitin content (g/100g of freeze-dried exoskeleton) measured in the exoskeleton from giant freshwater prawns fed the experimental diets over the 60-day trial.

Gene Expression
As evidenced in Figure 3, no significant differences were evident among the experimental groups in terms of jheh, chit3, hsp90, or α2m gene expression. However, catL was significantly upregulated in the HM3 and HM20 groups, compared with the HM0 group (Figure 3c).

Physical and Chemical Analyses
No significant differences were detected among the experimental groups considering pH, moisture muscle samples, and chitin content of exoskeleton samples from prawns fed the different experimental diets (Table 6). Table 6. pH and moisture (g/100g of muscle) measured in the muscle and chitin content (g/100g of freeze-dried exoskeleton) measured in the exoskeleton from giant freshwater prawns fed the experimental diets over the 60-day trial. Values are shown as mean ± SD (n = 4). The total lipid content and FA profile of muscle samples from prawns fed the different experimental diets are shown in Table 7. No significant differences were detected among the experimental groups in terms of total lipid, SFA, MUFA, and n3 PUFA contents. However, the n6 PUFA content was significantly higher in the HM3 and HM20 groups compared to the HM0 group (p < 0.001). Considering the FA profile, palmitic acid (C16:0) was the most represented SFA in all the experimental groups and its content was significantly higher in the HM0 group compared to the other groups (p < 0.001). Oleic acid (C18:1n9) was the most abundant MUFA in all the experimental groups, with significantly higher values in the HM0 and HM3 groups compared to the HM20 group (p < 0.001). Finally, considering PUFA, linoleic acid (C18:2n6; LA) and arachidonic acid (C20:4n6; ARA) contents were significantly higher in the HM3 group than in the other groups (p < 0.001). EPA (C20:5n3) content did not show significant differences among the experimental groups, while DHA (C22:6n3) content highlighted a progressive significant reduction from the HM0 group to the HM20 group (p = 0.00125).

Discussion
The use of HM as an alternative and more sustainable ingredient in aquafeed formulation has been widely studied in fish [63], but less in crustaceans. Several studies have been performed on the Pacific white shrimp (Litopenaeus vannamei) using different dietary inclusion levels of H. illucens larvae meal, obtaining promising results in terms of growth performances, antioxidant and immune response, digestive enzyme activity, intestinal microbiota, histomorphology of hepatopancreas, and muscle biochemical composition [64][65][66]. However, to the best of our knowledge, H. illucens has been scarcely explored as a possible alternative to FM in dietary formulations intended for a widely farmed Decapoda, such as the giant freshwater prawn [40]. The present study represents the first attempt to evaluate the replacement of FM and FO with spirulina-enriched full-fat HM on M. rosenbergii post-larvae reared in aquaponic systems to further improve aquaculture sustainability, promoting a zero-waste concept [67]. The three experimental diets tested in the present study, using aquaponic systems, resulted in comparable survival and growth rates, confirming the suitability of the diets and the farming technique for M. rosenbergii [42,68]. HM represents a good source of proteins and it was successfully used in the M. rosenbergii diet, which is known to require a relatively high dietary protein content (37% on DM) to support growth performances [69,70]. Accordingly, growth performances and morphological features were not negatively affected by the HM dietary inclusions, which additionally led to a higher HEW in prawns from the HM20 group. These results are in line with previous studies that demonstrated that a replacement of FM with H. illucens larvae meal up to 20% did not affect the growth performances of the Pacific white shrimp [64][65][66]71,72]. However, it should be pointed out that the use of high-protein diets in crustacean aquaculture generally causes a consequently high nitrogenous waste production that can interfere with water quality [73]. In this sense, as demonstrated by water quality analyses, aquaponic systems represented a valid method of exploiting this metabolic waste as a macronutrient for plant growth [3,42], avoiding economic losses due to prawn mortality linked to high ammonia levels and possible environmental issues [74,75].
The addition of spirulina in the insects' rearing substrate resulted in an increase in α-tocopherol from the HM3 to HM20 diets and in the presence of carotenoids in the HM20 diet, respectively, highlighting that this enrichment procedure can be a valid method to transfer these important antioxidant molecules from spirulina to H. illucens and, then, to the experimental diets. The provision of diets enriched with α-tocopherol and carotenoids has possibly reduced the secondary oxidative products in muscle samples from the HM0 group to the HM20 group, confirming the role of these molecules in preserving the muscle-quality traits [35][36][37]76].
However, the inclusion of full-fat HM in aquafeeds is usually associated with a dietary fatty acid profile alteration, especially regarding the SFA and the n3 PUFA contents [32,77]. Accordingly, the experimental diets tested here showed a slight increase in SFA content and a parallel wider decrease in n3 PUFA (especially in terms of EPA and DHA) from HM0 to HM20. Comparable amounts of n6 PUFA (largely constituted by LNA) were observed among the experimental diets. It has been demonstrated that the balance (more than the relative content) of dietary n3 and n6 PUFA is a crucial factor for the fatty acid requirement in prawns' nutrition [78]. In fact, despite the necessity to provide n3 PUFA [79], freshwater prawns such as M. rosenbergii might achieve the desired growth performance by relying on the dietary n6 PUFA [69]. This is a consequence of the terrestrial origin of the giant freshwater prawn's natural diet, rich in fatty acids from the n6 series [80]. The absence of a preference for either n3 or n6 series fatty acids to fulfil the PUFA requirements [81] sustained a proper growth of each experimental group. In addition, the tissue fatty acid profile of giant freshwater prawn generally reflects the dietary profile, due to the scarce ability of this crustacean to synthesize long-chain PUFA from shorter-chain precursors [82,83]. Accordingly, in the present study, the proportion among the fatty-acid classes in the diets reflected that of the muscle samples. The significantly higher amount of DHA in the muscle samples of prawns from the HM0 group emphasizes the importance of providing diets characterized by a proper amount of long-chain PUFA, in order to guarantee the quality of the final product and to meet the consumer's requirements. However, while the experimental diets showed a higher DHA content compared to EPA, an opposite result was obtained in muscle samples, reflecting the ability of M. rosenbergii to synthesize EPA from DHA and its bioconversion inability in the opposite direction [83].
The dietary lipid content can also affect the lipid deposition in hepatopancreas; in particular, the R cells are known to be the main site for lipid storage and are thus considered to be a crucial indicator of crustaceans' nutritional condition [84,85]. R cells are the most abundant cell type in the hepatopancreas, but nutritional stress can lead to a decrease in their prevalence in the tubules and vacuolation, with a consequent epithelium atrophy [84,86]. Accordingly, Wang et al. [87] evidenced vacuolar degeneration coupled with an alteration of the epithelium structure in Pacific white shrimp when the replacement of FM with H. illucens larvae meal exceeded 60%. However, in the present study, which used lower replacement percentages, epithelium height and lipid storage were not affected by the provision of HM diets, as demonstrated by the comparable frequency of R cells and the high abundance of lipid droplets in all the experimental groups.
Dietary nutrients are absorbed and stored by the R cells to be subsequently mobilized to sustain energy-demanding processes such as molting [88]. The massive presence of lipid droplets in R cells in all the experimental groups reflected a proper nutrient provision by the experimental diets and evidenced the necessity of prawns to store energy reserves during the inter-molt stage [83]. Crustaceans periodically degrade the old exoskeleton in favor of a newly synthesized one (pre-molt phase) and, during molting, they face a rapid uptake of water that increases the animal size. After a post-molt phase, in which the hardness of the new exoskeleton is improved, the water absorbed during molting is gradually replaced by tissue growth and reserves are stored (inter-molt phase) [83]. This process is under hormonal control. In particular, methyl farnesoate is the crustacean analogue of the pleiotropic juvenile hormone (JH) that is typical of insects and, like JH, it is involved in several processes including molting [89]. This hormone, together with the ecdysone, gradually increases in the pre-molt phase, reaching the maximum levels near molting [90,91]. However, a key determinant for successful molting is the rapid decrease in methyl farnesoate due to its JHEH-mediated inactivation. It has been demonstrated that in M. rosenbergii, the higher jheh expression in the pre-molt stage led to a successful molt in a short time [92]. The crustacean hepatopancreas is considered the main site for methyl farnesoate inactivation due to the highest level of jheh expression [92]. In the present study, the absence of significant differences in jheh gene expression represented further confirmation that the molting cycle (and thus somatic growth) and its regulation were not affected by the different dietary treatments.
The activity of digestive enzymes can have a direct effect in supporting molting and growth because of the breakage of the dietary high-weight biomolecules into more available forms required for metabolism or storage [38]. In this regard, the primary role of the hepatopancreas is the digestive enzymes' synthesis and secretion, the digestion of the ingested food, and the subsequent nutrients' uptake [93]. In particular, B cells produce digestive enzymes that are involved in intracellular digestion and concentrate in the vacuole the absorbed materials, and secrete them in the tubular lumen at the end of the digestive process for reabsorption by R cells [88]. In the present study, a higher relative abundance of B cells was detected in the HM3 and HM20 groups than in the HM0 group. This result was fully supported by the expression of catL which codifies for the cathepsin L, a protease stored in the digestive vacuole of B cells and secreted into the tubular lumen of the hepatopancreas for extracellular digestion [94]. In this regard, it should be pointed out that Hu et al. [94] detected catL mRNA only in F cells but not in mature B cells that only host the active enzyme. However, several authors suggested that F cells are the precursors of B cells, and from an F cell to a B cell there is a continuum of transition (referred to as F/B cells) [95]. Therefore, although we only measured the mRNA relative abundance of catL, this result supports the abundance of B cells due to the fast synthesis of cathepsin L and the quick transition from F cell type to B cell type [94].
An increased B cells population in the hepatopancreas is usually associated with the reduced bioavailability of dietary nutrients [96,97]. Several studies on fish reported that one of the possible drawbacks related to the dietary inclusion of HM can be attributed to chitin, which, due to its complex matrix, can impair nutrient absorption, creating a barrier on the absorptive epithelium [98][99][100]. In addition, Kumar et al. [101] found that M. rosenbergii that were fed post-larvae with 10% purified chitin had a lower growth rate than those fed with 5% purified chitin, confirming that in crustaceans this complex molecule can act as a barrier against nutrient absorption. However, crustaceans possess specific chitinases expressed in the hepatopancreas, among which is the CHIT3 that is involved in the degradation of chitin-containing feeds; chitinases are part of the natural diet of this species [83,102]. In the present study, the chit3 gene expression in the hepatopancreas did not significantly vary among the experimental groups, highlighting that HM diets did not enhance the production of this chitinase. For that reason, instead of a nutrient absorption impairment caused by chitin, the increased B cells number observed in the HM3 and HM20 groups could be related to the different compositions of the lipid fraction among the experimental diets. In fact, B cells may increase in number and size in response to high dietary lipids, producing and recycling fat emulsifiers to counteract their hydrophobic features that slow down the digestive processes and to facilitate their endocytosis [103,104]. As the diets were iso-lipidic, this conclusion could possibly be related to the dietary amount of SFA and MUFA that increased with the increasing dietary inclusion levels of HM and to the ability of M. rosenbergii to digest and use these fatty-acid classes as an energy source [80,83].
Finally, the hepatopancreas, in addition to being a sensitive indicator for the nutritional status, is used to monitor the health conditions of crustaceans, as it is involved in the immune and stress response [96,105,106]. Crustaceans only possess innate immunity due to the absence of cells from the myeloid lineage that are able to produce specific antibodies to face repetitive infections [107]. Specifically, crustaceans have a rapid innate immunity consisting of various prophenoloxidase (proPO) system immune-related proteins, including α2-macroglobulin, which acts against pathogens and regulates the proPO activation [108]. In addition, it has been demonstrated that the chitin-related genes expressed in the hepatopancreas of crustaceans may be involved in the immune response and disease resistance through direct or indirect regulation [109,110]. The expression of the immune response marker a2m, supported by the absence of significant differences in terms of chit3 gene expression, confirmed that the experimental diets used in the present study did not affect the health status of M. rosenbergii. This conclusion is further supported by the expression of hsp90, a stress-related biomarker that can also play a significant role in host immunity and the health of crustaceans [111].

Conclusions
The experimental diets used in the present study properly sustained survival and growth and fulfilled the PUFA requirement of giant freshwater prawn post-larvae. The dietary lipid content of each experimental diet led to a massive presence of lipid droplets in R cells in all the experimental groups, allowing a proper storage of energy sources to sustain molting processes. However, the diverse composition of the lipid fraction among the experimental diets resulted in an increase in B cells abundance in the HM3 and HM20 groups. In addition, the use of spirulina-enriched HM in the diets allowed the transfer of important bioactive molecules to the prawns, which possibly played a crucial role in preserving muscle-quality traits. Finally, the hepatopancreas health status was preserved in all the experimental groups, as evidenced by the histological analyses and the expression of immune and stress markers.
The present study represents an example of how M. rosenbergii culture can be implemented with more sustainable aquafeeds and farming techniques such as aquaponic systems, to promote both the valorization of the circular economy and the zero-waste concepts in aquaculture.  Informed Consent Statement: Not applicable, as this research did not involve humans.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.